Process for production of inosine and 5&#39;-guanylic acid nucleotides

ABSTRACT

A process for producing inosine and 5&#39;&#39;-guanylic acid nucleotides by fermentation which comprises culturing a mutant strain having the ability to accumulate inosine and simultaneously to convert 5&#39;&#39;-xanthylic acid into 5&#39;&#39;-guanylic acid nucleotides under aerobic conditions in an aqueous nutrient medium containing 5&#39;&#39;-xanthylic acid. Strains advantageously employed are Brevibacterium ammoniagenes ATCC 21264 and Corynebacterium glutamicum ATCC 21266.

United States Patent [72] Inventors Shigeo Abe Tokyo; Akira Furuya,Machida-shi; Ryo Okachi, Machlda-shi, all of Japan [21] Appl. No.758,591

[22] Filed Sept. 9, 1968 [45] Patented Oct. 26, 1971 [73] Assignee KyowaHakko Kogyo Co., Ltd.

Tokyo, Japan [32] Priority Sept. 27, 1967 [3 3 Japan [54] PROCESS FORPRODUCTION OF INOSlNE AND 5'- GUANYLIC ACID NUCLEOTIDES 15 Claims, NoDrawings [52] US. CL... [51] Int. Cl C12d 13/06 [50] Field of Search195/28 N [56] References Cited UNITED STATES PATENTS 3,296,087 1/1967Mitsugi et al. 195/28 N Primary Examiner-Alvin E. TanenholtzAttorney-Craig, Antonelli, Stewart & Hill ABSTRACT: A process forproducing inosine and 5'-guanylic acid nucleotides by fermentation whichcomprises culturing a mutant strain having the ability to accumulateinosine and simultaneously to convert 5-xanthy1ic acid into 5'-guanylicacid nucleotides under aerobic conditions in an aqueous nutrient mediumcontaining 5-xanthylic acid. Strains advantageously employed areBrevibaclerium ammoniagenes ATCC 21264 and Carynebaclerium glutamicumATCC 21266.

The present invention relates to a process for producing inosine and'-guanylic acid system nucleotides. More particularly, it relates to aprocess for the production of inosine and 5'-guanylic acid nucleotidesby fermentation. Even more particularly, the invention relates to aprocess for the production of inosine and 5'-guanylic acid nucleotidessuch as 5- guanosine-l-phosphoric acid (5'-GDP), and 5 '-guanosine-2-phosphoric acid (5'-GDP) and 5 '-guanosine-3-phosphoric acid (5'-GTP) byfermentation with micro-organisms having particular characteristics.

inosine, which is hypoxanthine riboside, is found in meat and meatextracts and in sugar beets. inosine as well as the 5 guanylic acidnucleotides have found wide uses and applications in the biochemicalfield.

One of the objects of the present invention is to provide an improvedprocess for the production of inosine and 5-guanylin acid nucleotideswhich overcomes the disadvantages and deficiencies of the prior artmethods.

Another object of the present invention is to provide a process forproducing inosine and 5-guanylic acid nucleotides by fermentation withparticular micro-organisms which may be carried out in an efficaciousand simple manner.

A further object of the invention is to provide a process for producingsaid products by fermentation which may be carried out advantageouslyand economically on an industrial scale to give a high yield of product.

A still further object of the invention is to provide inosine and'5'-guanylic acid system nucleotides such as 5'-guanosinel-phosphoricacid, 5-guanosine-2-phosphoric acid and 5" guanosine-Ii-phosphoric acid.

These and other objects and advantages of the present in vention willbecome apparent to those skilled in the art from a consideration of thefollowing specification and claims.

in accordance with the present invention, it has been found thatcultivating or culturing a micro-organism having the ability to produceinosine and a capacity for converting 5 '-xanthylic acid into 5-guanylicacid nucleotides with a high yield in a culture medium containing5"xanthylic acid makes it possible to accumulate inosine and tosimultaneously convert 5'- xanthylic acid into S-guanylic acidnucleotides. The products may then be recovered as desired from thefermentation liquor in high yield.

The present invention has resulted from many years of study by theinventors on the production of nucleotides and nucleosides byfermentation and the discovery of micro-organism mutants having theability to accumulate inosine and the capacity for converting5'-xanthylic acid into 5'-guanylic acid nucleotides in high yield.Micro-organism strains employed in the present invention are mutantsobtained by subjecting micro-organisms belonging to various genera tothe application of ultraviolet rays or cobalt 60-y-rays or to chemicaltreatment with nitrous acid, dimethyl sulfate, or nitrosoguanidine. Theresulting mutants are characterized in requiring adenine compounds(adenine, adenosine or adenylic acid) for their growth or that thegrowth thereof is accelerated by purine compounds. As noted above, thesemutants cause the accumulation of large amounts of inosine in theculture liquor. Moreover, the mutants possess the property of convertingS'ctanthylic acid, which is added to the culture medium, into5'-guanylic acid nucleotides in high yield. Mutants having othernutrient-requiring properties such as, for example, amino acids,vitamins, purines, pyrimidines and the like, as well as the abovecharacteristics, are also employable in the process of the presentinvention.

Preferred strains to be employed in the present invention includeBrevibacterium ammoniagenes iXG-Zl ATTC 21264 and Corynebacreriumglutamicum iXG-3l ATCC 2l266.

Either a synthetic culture medium or a natural nutrient medium issuitable for the cultivation as long as it contains the essentialnutrients for the growth of the micro-organism strain employed. Suchnutrients are well known in the art and include substances such as acarbon source, a nitrogen source,

inorganic compounds and the like which are utilized by themicro-organism employed in appropriate amounts. Exemplary of carbonsources which may be employed in the aqueous nutrient medium arecarbohydrates such as glucose, fructose, maltose, sucrose, starch,starch hydrolysate, waste molasses, etc., or any other suitable carbonsource such as glycerol, mannitol, sorbitol, organic acids such asacetic acid, lactic acid, glutamic acid, etc. These substances may beused either singly or in mixtures of two or more. .As a nitrogen source,various kinds of inorganic or organic salts or compounds, such as urea,ammonia, or ammonium salts such as ammonium chloride, ammonium sulfate,ammonium nitrate, ammonium phosphate, etc., or natural substancescontaining nitrogen, such as cornsteep liquor, yeast extract, meatextract, fishmeal, peptone, bouillon, casein hydrolysatcs, fishsolubles, rice bran extract, casamino acid, etc. may be employed. Thesesubstances may also be used either singly or in combinations of two ormore. Inorganic compounds which may be added to the culture mediuminclude magnesium sulfate, sodium phosphate, potassium dihydrogenphosphate, potassium monohydrogen phosphate, iron sulfate, manganesechloride, calcium chloride, sodium chloride, einc sulfate, etc.Moreover, it may also be necessary to add certain essential nutrients tothe culture medium, depending upon the particular micro-organismemployed, such as amino acids and/or vitamins, for example, biotin,thiamine, cohalemin and the like, as well as adenine compounds as notedabove.

it is sometimes advantageous to add u surfaceactive agent to the culturemedium. Surface-active agents which may be employed may be eitheranionic, cationic or nonionic. Such agents are well known in the artand, generically, comprise substances such as the sodium salts of highmolecular weight alkyl sulfates or sulfonates, polyoxyethyiene glycolderivatives, higher fatty acids having from 12 to 20 carbon atoms, forexample, lauric acid, myristic acid, palmitic acid, stearic acid, oleicacid and the like, organic esters or" higher fatty acids, such assorbitan monooleate, etc. Specific examples of surface-active agentswhich may be employed are to be found in, for example, copendingapplication Ser. No. 643,832, filed on June 6, 1967, the disclosure ofwhich is herein expressly incorporated by reference.

Pursuant to the present invention, a mutant strain having the ability toaccumulate inosine and simultaneously to convert 5'-xanthylic acid,added to the medium, into 5'-guanylic acid nucleotides is cultivated inan aqueous nutrient medium. The 5-xanthylic acid may be added to themedium at the time when the micro-organism is inoculated therein, or itmay be added after some of the growth of the micro-organism has takenplace. The amount of 5'-xanthylic acid added to the medium generallyranges from about 5.0 to 30.0 mg./ml., but it is to be understood thatthe concentration thereof added to the medium will vary depending uponthe particular strain employed. Very pure 5 '-xanthylic acid itself, orS'oranthylic acidcontaining substances as well as culture liquorscontaining 5'- xanthylic acid (prepared by fermentation), may be used asthe additive to the medium, so long as the particular form of xanthylicacid employed does not adversely affect the growth of the microorganism,the accumulation of inosine or the conversion into 5-guanylic acidnucleotides.

The fermentation is conducted under aerobic conditions, such as aerobicshaking of the culture or with aeration and agitation of a submergedculture, at a temperature of, for example, about 20" to 40 C. and at apli of about, for example, 5.0 to 9.0 The pli of the medium may beadjusted appropriately with hydrochloric acid, sulfuric acid, phosphoricacid, urea, aqueous ammonia, sodium hydroxide, potassium hydroxide etc.,in accordance with the strain used, during the cultivation procedure.After about 2 to 6 days of culturing under these conditions, largequantities of 5'-guanylic acid nucleotides or a mixture of 5-guanylicacid nucleotides together with inosine are accumulated in the cultureliquor and the micro-organism cells. After the completion of culturing,the accumulated products can be recovered as single, in-

The following examples are given merely as illustrative of 5 the presentinvention and are not to be considered as limiting. Unless otherwisenoted, the percentages therein are by weight per liter of water.

EXAMPLE 1 Brevibacterium ammaniagenes iXG-2l ATCC 21264, which isobtained from Brevibacterium ammoniagenes ATCC 6872 by irradiation withultraviolet rays, is employed as the seed strain. It requires adeninefor its growth. The mutant is subjected to culturing with aerobicshaking at 30 C. for 24 hours in a culture medium (pH 7.2) containing 2percent of glucose, 1 percent of peptone, l percent of yeast extract and0.3 percent of sodium chloride in order to obtain a seed culture.

20-ml. portions of the seed medium or the fermentation medium describedbelow are poured into conical flasks of 250-ml. volume and aresterilized at 120 C. for 10 minutes under an elevated pressure.

A fennentation medium having the following composition is prepared:

10% glucose 0.6% urea 1.0% KJIPO 1.0% MgS0,-7,0

30p.g./l biotin 20mg./l adenine 5mg./l vitamin B,

lmg./l calcium pantothenate 100% meat extract The pH value of thefermentation medium is adjusted to 7.8 by the use of dilute sodiumhydroxide before sterilization.

Before inoculating-the seed culture into the fermentation medium,-xanthylic acid (80 percent purity) is added to the fermentation mediumin an amount necessary to give a concentration of 20 mg./ml. The seedculture is inoculated into a fermentation medium in the amount ofpercent by volume, based on the amount of fermentation medium.

Culturing is then carried out with aerobic shaking of the culture at 30C. After 72 hours of culturing, the pH value of the liquor is adjustedto 7.5 with dilute ammonia water. The fermentation is completed after120 hours of culturing.

After the completion of fermentation, l3 mg./ml. of inosine, 3.4 mg./ml.of5'-GMP, 2.8 mg./ml. of5-GDP and 7.7 mg./ml. of 5-GTP are found to beaccumulated in the culture liquor. In addition, the accumulation ofsmall amounts of guanosine, guanine and hypoxanthine, respectively, isobserved. A small amount of residual 5-xanthylic acid remains in themedium.

EXAMPLE 2 Brevibacterium ammaniagenes iXG-Zl ATCC 21264 is used as theseed micro-organism. The seed strain is cultured in the same manner asdescribed in Example 1 in order to obtain a seed culture.

The fermentation medium employed has the following composition:

13% starch-saccharified liquor (calculated as glucose) 0.6% urea 0.5%K,HPO

5 mg./l vitamin B,

10 mg./l calcium pantothenate 30 mg./l adenine 30 tag/l biotin 1.0%casamino acid The seed culture liquor in an amount of 10 percent byvolume is inoculated into the fermentation medium. Culturing is then outwith aerobic shaking of the culture at 35 C. for 72 hours. At thispoint, an equal amount of 5'-xanthylic acid-containing fermentationliquor (containing 30 mg./ml. of 5- xanthylic acid) is added to theculture liquor. In addition, 3.0 mg./ml. of Nimean S-215 (manufacturedby Nippon Yushi Co. Ltd.), a surface-active agent, is added to theliquor. Culturing is then continued for a further 48 hours. During theculturing an ammonium sulfate solution is added in an amount necessaryto give a final concentration of 0.5 percent. Thereafter, the pH valueis adjusted to 7.8 with dilute sodium hydroxide.

After the completion of fermentation, 6.8 mg./ml. of inosine, 7.4mg./ml. of 5'-GMP and 3.4 mg./ml. of 5'-GTP are accumulated in theliquor. Moreover, the accumulation of small amounts of 5'-GDP, guanineand guanosine, respectively, is observed. Only a small amount ofresidual 5'-xanthylic acid remains in the culture liquor.

EXAMPLE 3 Corynebacterium glutamicum iXG-3l ATCC 21266, a mutant strainobtained by treating Corynebacterium gluramicum ATCC 13032 withnitrosoguanidine, is employed as the seed strain. The characteristic ofthis strain is that its growth is considerably accelerated by adenine,guanine or hypoxanthine.

A fermentation medium having the following composition is employed:

13% glucose 1.0% KgHOP4 1.0% M som-no 1.5% NH,cl.

0.5% yeast extract 3% CaCO,

The CaCO is added after sterilization. The pH of the medium is 7.3.

The seed culture is inoculated into the fermentation medium in theamount of 10 percent by volume. Moreover, 20 mg./ml. of 5'-xanthylicacid is added to the medium at the time of inoculation. The otherconditions employed are the same as described in Example 1. Culturing iscarried out with aerobic shaking for hours. During culturing, a 20percent urea solution is used for adjusting the pH value.

As a result of the fermentation, the accumulation of 9.7 mg./ml. ofinosine, 6.4 mg./ml. of 5'-GMP, 4.2 mg./ml. of 5'- GDP and 3.8 mg./ml.of 5-GTP, respectively, is observed in the culture liquor. In addition,the presence of a small amount of remaining 5 '-xanthylic acid is foundin the liquor.

The invention being thus described, it will be obvious that the same maybe varied in many ways. Such variations are not to be regarded as adeparture from the spirit and scope of the invention, and all suchmodifications are intended to be included within the scope of thefollowing claims.

We claim:

1. A process for producing inosine and 5-gyanylic acid nucleotides whichcomprises culturing a micro-organism belonging to Brevibacteriumammoniagenes or Corynebacrerium glutamicum, said micro-organism beingcapable of producing inosine and having the capacity of converting5'-xanthylic acid into 5'-guanylic acid nucleotides, under aerobicconditions in an aqueous nutrient medium containing 5'-xanthylic acid,accumulating both inosine and the 5-guanylic acid nucleotides in theresultant culture liquor, and recovering said inosine and 5 '-guanylicacid nucleotides therefrom.

2. The process of claim 1, wherein culturing is carried out at atemperature of about 20 to 40 C. and at a pH of about 5 to 9 3. Theprocess of claim 1, wherein said 5'-guanylic acid nucleotides areselected from the group consisting of 5- guanosine-l-phosphoric acid,5'-guanosine-2-phosphoric acid and 5'-guanosine-3-phosphoric acid.

4. The process of claim 1, wherein said nutrient medium also contains asurface-active agent.

5. The process of claim 1, wherein the 5-xanthylic acid is added to themedium at the initiation of culturing.

6. The process of claim 1, wherein the 5'-xanthylic acid is added to themedium after the initiation of culturing and during the culturingperiod.

7. The process of claim 1, wherein the amount of 5- xanthylic acidpresent in the medium is about 5.0 to 30.0 mg./ml.

8. The process of claini 1, wherein a culture liquor containing 5'-xanthylic acid is added to said medium.

9. The process of claim 2, wherein said microorganism is Brevibacteriumammoniagenes ATCC 21264.

10. The process of claim 2, wherein said microorganism isCorynebacterium glutamicum ATCC 21266.

ll. A process for producing inosine and 5'-guanylic acid nucleotideswhich comprises culturing a micro-organism selected from the groupconsisting of Brevibacterium ammoniagenes ATCC 21264 and Corynebacleriumgluramicum ATCC 21266 under aerobic conditions in an aqueous nutrientmedium containing 5'-xanthylic acid, accumulating both inosine and the5'-guanylic acid nucleotides in the resultant culture liquor, andrecovering said inosine and 5-guanylic acid nucleotides therefrom.

12. The process of claim 11, wherein culturing is carried out at atemperature of about 20 to 40 C. and at a pH of about 5 to 9.

13. The process of claim 12, wherein said 5'-guanylic acid nucleotidesare selected from the group consisting of 5'- guanosine-l-phosphoricacid, 5'-guanosine-2-phosphoric acid and 5 '-guanosine 3-phosphoricacid.

14. The process of claim 13, where said nutrient medium also contains asurface-active agent.

15. The process of claim 13, wherein the amount of 5'- xanthylic acidpresent in the medium: is about 5.0 to 30.0 mg./ml.

2. The process of claim 1, wherein culturing is carried out at atemperature of about 20* to 40* C. and at a pH of about 5 to
 9. 3. Theprocess of claim 1, wherein said 5''-guanylic acid nucleotides areselected from the group consisting of 5''-guanosine-1-phosphoric acid,5''-guanosine-2-phosphoric acid and 5''-guanosine-3-phosphoric acid. 4.The process of claim 1, wherein said nutrient medium also contains asurface-active agent.
 5. The process of claim 1, wherein the5''-xanthylic acid is added to the medium at the initiation ofculturing.
 6. The process of claim 1, wherein the 5''-xanthylic acid isadded to the medium after the initiation of culturing and during theculturing period.
 7. The process of claim 1, wherein the amount of5''-xanthylic acid present in the medium is about 5.0 to 30.0 mg./ml. 8.The process of claim 1, wherein a culture liquor containing5''-xanthylic acid is added to said medium.
 9. The process of claim 2,wherein said micro-organism is Brevibacterium ammoniagenes ATCC 21264.10. The process of claim 2, wherein said micro-organism isCorynebacterium glutamicum ATCC
 21266. 11. A process for producinginosine and 5''-guanylic acid nucleotides which comprises culturing amicro-organism selected from the group consisting of Brevibacteriumammoniagenes ATCC 21264 and Corynebacterium glutamicum ATCC 21266 underaerobic conditions in an aqueous nutrient medium containing5''-xanthylic acid, accumulating both inosine and the 5''-guanylic acidnucleotides in the resultant culture liquor, and recovering said inosineand 5''-guanylic acid nucleotides therefrom.
 12. The process of claim11, wherein culturing is carried out at a temperature of about 20* to40* C. and at a pH of about 5 to
 9. 13. The process of claim 12, whereinsaid 5''-guanylic acid nucleotides are selected from the groupconsisting of 5''-guanosine-1-phosphoric acid,5''-guanosine-2-phosphoric acid and 5''-guanosine-3-phosphoric acid. 14.The process of claim 13, where said nutrient medium also contains asurface-active agent.
 15. The process of claim 13, wherein the amount of5''-xanthylic acid present in the medium is about 5.0 to 30.0 mg./ml.